Scanning apparatus and methods useful for detection of chemical and biological analytes

ABSTRACT

An apparatus can include a vessel, a reference surface, a preload, a scan actuator, and a transmitter. The reference surface can form a structural loop with a detector. The preload can be configured to urge the vessel to contact an area on the reference surface. The scan actuator can be configured to slide the vessel along the reference surface in a scan dimension. The transmitter can be configured to direct signal from the vessel to a detector and/or direct energy from an energy source to the vessel, when the vessel is urged by the preload to contact the reference surface.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of Ser. No. 15/998,727filed Aug. 15, 2018, entitled “SCANNING APPARATUS AND METHODS USEFUL FORDETECTION OF CHEMICAL AND BIOLOGICAL ANALYTES,” which claims priority toU.S. Provisional Application No. 62/545,606 filed on Aug. 15, 2017 andentitled “SCANNING APPARATUS AND METHODS USEFUL FOR DETECTION OFCHEMICAL AND BIOLOGICAL ANALYTES,” the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND

The present disclosure relates generally to detection of chemical andbiological analytes and has specific applicability to nucleic acidsequencing.

The determination of nucleic acid sequence information is important inbiological and medical research. Sequence information is used foridentifying gene associations with diseases and phenotypes, identifyingpotential drug targets, and understanding the mechanisms of diseasedevelopment and progress. Sequence information is an important part ofpersonalized medicine, where it can be used to optimize the diagnosis,treatment, or prevention of disease for a specific individual.

Many scientists and medical practitioners struggle to tap into modernsequencing technology due to prohibitive costs to run and maintaincomplex instrumentation in current commercial offerings. These platformsfavor centralized laboratories in which expensive “factory scale”instruments are run by highly trained specialists, and samples arebatched to achieve economies of scale. This centralized system offersvery little flexibility in terms of performance specifications—users areforced into ecosystems that are unnecessarily limited in scope andvariety of use. When it comes to clinical applications, the centralizedmodel is costly for doctors and their patients in terms of both the timeand money required to ship patient samples from local clinics to distantsequencing labs. Further delays can be incurred as a centralizedsequencing lab waits to receive sufficient number of samples to batchtogether into an economical run. Other applied markets such asforensics, veterinary diagnostics, food safety, agricultural analysisand environmental analysis suffer similar limitations.

Thus, there is a need for a sequencing platform that is better suitedfor use in local laboratories in support of a decentralized system ofresearch and clinical care. The present invention satisfies this needand provides related advantages as well.

BRIEF SUMMARY

The present disclosure provides a detection apparatus that can include(a) a vessel having a lumen and a wall, wherein the wall has an internalsurface and an external surface, wherein the internal surface contactsthe lumen; (b) a reference surface that forms a structural loop with adetector; (c) a preload configured to urge the external surface of thevessel to contact an area on the reference surface; (d) a scan actuatorconfigured to slide the vessel along the reference surface in a scandimension; and (e) a transmitter configured to direct, to the detector,a signal from the internal surface or the lumen, when the externalsurface of the vessel is urged by the preload to contact the referencesurface.

Also provided is a method of scanning a vessel. The method can include(a) translating a vessel along a reference surface of a detectionapparatus, wherein the vessel comprises a lumen and a wall, wherein thelumen comprises analytes, wherein the reference surface contacts atleast a portion of the vessel during the translating, and wherein thereference surface forms a structural loop with a detector; and (b)detecting the analytes at different locations along the vessel using thedetector, wherein the vessel is urged to the reference surface by apreload during the detecting, thereby scanning the vessel.

In some embodiments, a method of scanning a vessel can include (a)examining a first subset of analytes in a vessel while applying apreload to a first portion of the vessel, wherein the preload positionsthe first subset of analytes to occupy an xy plane in a detection zone,wherein the preload is not applied to a second portion of the vessel;(b) translating the vessel to position a second subset of the analytesin the xy plane of the detection zone; and (c) examining the secondsubset of the analytes in the vessel while applying the preload to asecond portion of the vessel, wherein the preload positions the secondsubset of the analytes to occupy the xy plane of the detection zone,wherein the preload is not applied to the first portion of the vessel,thereby scanning the vessel.

The present disclosure provides reactor apparatus. A reactor apparatuscan include (a) a vessel having a lumen and a wall, wherein the wall hasan internal surface and an external surface, wherein the internalsurface contacts the lumen; (b) a reference surface that forms astructural loop with an energy source; (c) a preload configured to urgethe external surface of the vessel to contact an area on the referencesurface; (d) a scan actuator configured to slide the vessel along thereference surface in a scan dimension; and (e) a transmitter configuredto direct energy from the energy source to the internal surface or thelumen when the external surface of the vessel is urged by the preload tocontact the reference surface.

Also provided is a method of performing reactions in a vessel. Themethod can include (a) translating a vessel along a reference surface ofa reactor apparatus, wherein the vessel comprises a lumen and a wall,wherein the lumen comprises reactants, wherein the reference surfacecontacts at least a portion of the vessel during the translating, andwherein the reference surface forms a structural loop with an energysource; and (b) directing energy from the energy source to the reactantsat different locations along the vessel, wherein the vessel is urged tothe reference surface by a preload during the directing of the energy tothe reactants, thereby performing reactions in the vessel.

A method of performing reactions in a vessel can include (a) deliveringenergy from a reactor apparatus to a first subset of reactants in avessel while applying a preload to a first portion of the vessel,wherein the preload positions the first subset of reactants to occupy anxy plane of a reaction zone, wherein the preload is not applied to asecond portion of the vessel; (b) translating the vessel to position asecond subset of the reactants in the xy plane of the reaction zone; and(c) delivering energy from the reactor apparatus to the second subset ofthe analytes in the vessel while applying the preload to a secondportion of the vessel, wherein the preload positions the second subsetof the analytes to occupy the xy plane, wherein the preload is notapplied to the first portion of the vessel, thereby performing reactionsin the vessel.

In particular embodiments, the present disclosure provides a detectionapparatus that includes (a) a vessel having a lumen and a wall, whereinthe wall has an internal surface and an external surface, wherein theinternal surface contacts the lumen, and wherein the external surfacehas length l in a scan dimension x; (b) a reference surface; (c) apreload configured to urge the external surface of the vessel to contactan area on the reference surface, optionally the area of contact canhave a maximum length in the scan dimension x that is shorter thanlength l; (d) a scan actuator configured to slide the vessel along thereference surface in the scan dimension x; (e) a detector; and (f) anobjective configured to direct radiation from the vessel to the detectorwhen the external surface of the vessel is urged by the preload tocontact the reference surface.

Also provided is a method of optically scanning a vessel. The method caninclude (a) providing a vessel having a lumen and a wall, wherein thelumen contains optically detectable analytes and wherein the wall istransparent to the optically detectable analytes; (b) translating alength of the vessel along a reference surface and detecting theoptically detectable analytes at different locations along the length,wherein the reference surface contacts only a portion of the length ofthe vessel at any time during the translation, wherein the vessel isurged to the reference surface by a preload during the detection,wherein the detection includes transmitting radiation through the wall,then through an objective and then to a detector, thereby opticallyscanning the vessel.

The present disclosure further provides a detection apparatus thatincludes (a) a vessel having a lumen and a wall, wherein the wall has aninternal surface and an external surface, wherein the wall has aplurality of discrete contacts between the internal surface and theexternal surface, wherein the internal surface contacts the lumen, andwherein the plurality of discrete contacts occupies a length l in a scandimension x; (b) a transmissive surface; (c) a preload configured tourge discrete contacts on the external surface of the vessel to contactthe transmissive surface, optionally the area of the transmissivesurface can have a maximum length in the scan dimension x that isshorter than length l; (d) a scan actuator configured to slide thevessel along the transmissive surface in the scan dimension x; and (e) adetector configured to acquire signals from the discrete contacts viathe transmissive surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows dimensions and axes of rotation used to describe relativeorientation of components in optical systems and other apparatus setforth herein.

FIG. 2A shows an exploded, profile view of a flow cell and detectionapparatus;

FIG. 2B shows a profile view of the flow cell in contact with adetection apparatus;

FIG. 2C shows a perspective view of the flow cell in contact with thedetection apparatus; and FIG. 2D shows an exploded, perspective view ofthe flow cell in contact with the detection apparatus.

FIG. 3A and FIG. 3B show front and rear perspective views of a filmsprocket mechanism for translating a flow cell relative to a detectionapparatus.

FIG. 4A shows a flow cell cartridge; FIG. 4B shows a film sprocket andguide interacting with the flow cell cartridge; FIG. 4C shows a flowcell; and FIG. 4D shows a perspective view of the film sprocket, guide,flow cell cartridge, flow cell and a motor for the film sprocket.

FIG. 5A and FIG. 5B show front and rear perspective views of a spur gearmechanism for translating a flow cell relative to a detection apparatus.

FIG. 6A and FIG. 6B show front and rear perspective views of a ballscrew mechanism for translating a flow cell relative to a detectionapparatus.

FIG. 7A shows a perspective view of a heating plate and film sprocketscanning mechanism and FIG. 7B shows a perspective view of an objectiveand the heating plate and film sprocket scanning mechanism.

FIG. 8A shows a perspective view of a fluidic caddy with an attachedflow cell;

FIG. 8B shows an expanded view of the attachment points for the flowcell to the caddy; FIG. 8C shows a front view of the fluidic caddy withattached flow cell; FIG. 8D shows a side view of the fluidic caddy withattached flow cell; FIG. 8E shows a top view of the fluidic caddy withattached flow cell; and FIG. 8F shows a perspective view of the fluidiccaddy emptied of several fluidic components.

FIG. 9A shows a perspective view of a fluidic caddy and flow cellinteracting with a detection apparatus; FIG. 9B shows a top view of thefluidic caddy and flow cell interacting with the detection apparatus;and FIG. 9C shows a perspective view of the fluidic caddy disengagedfrom the detection apparatus.

FIG. 10A shows a side view, and expanded view of section c, for afluidic caddy with attached flow cell; FIG. 10B shows the expanded viewof the flow cell after being released from the fluidic caddy; FIG. 10Cshows a top view of a fluidic caddy engaged with components of adetection apparatus; FIG. 10D shows a cutaway view of the fluidic caddy(along line m) engaged with components of a detection apparatus; andFIG. 10E shows an expanded view of the fluidic caddy engaged withcomponents of a detection apparatus.

FIG. 11 shows a cutaway profile view of a rigid support aligned to aflow cell and an immersion objective.

DETAILED DESCRIPTION

The present disclosure provides apparatus and methods for detectinganalytes, such as chemical or biological analytes. The detection canoccur for analytes that are consumed, modified or produced as part of areaction of interest. Several embodiments of the apparatus and methodsare well suited to detection of repetitive reactions such as those usedto characterize or synthesize polymers. A wide variety of polymers existin nature and an infinite variety of polymers can be made by naturalprocesses, or synthetic processes that nevertheless utilize a relativelysmall number of monomeric building blocks. For example, DNA issynthesized in nature from four different nucleotides, as is RNA.Protein, another ubiquitous polymer, is made from 20 differentgenetically encoded amino acids. Apparatus and methods of the presentdisclosure can be configured to sequentially detect monomeric buildingblocks, thereby providing a capability to identify any sequence. Inparticular embodiments, the apparatus and methods can be configured todetect analytes that are consumed, produced or modified during amulti-cycle, repetitive reaction process. For example, intermediateproducts can be detected at each individual cycle. By way of morespecific example, nucleic acids can be sequenced by serially deliveringreagents that specifically react with, or bind to, the four differenttypes of nucleotide monomers, and components of each reaction (e.g.labeled nucleotides or labeled polymerases) can be detected during orafter each cycle. Alternatively, nucleic acids can be synthesized byserially delivering one of four different nucleotide monomers, orprecursors thereof, in a predefined order to a growing polymer and thenproducts (e.g. blocking moieties released during deprotection) can bedetected for each cycle. Sequencing or synthesis of proteins can also bedetected cyclically using apparatus and methods set forth herein.

Various aspects of the present invention are exemplified with regard toscanning detection. It will be understood that apparatus and methods setforth herein can be used for precise spatially resolved manipulation ofreagents or substrates in a vessel whether or not the reagents orsubstrates are detected. For example, light energy can be delivered to avessel to perform photoreactions at spatially resolved locations in avessel or to fabricate light responsive materials in a spatiallyresolved manner.

This disclosure provides apparatus and methods that can be used toobserve a vessel by translational movement of the vessel relative to adetector. Also provided are apparatus and methods to address a vessel,for example, by delivery of localized energy, by translational movementof the vessel relative to an energy source. When detecting analytes,this scanning motion allows the detector to collect signals fromsequential subsections of the vessel. The collective combination ofsignals sums to a total field of detection that is larger than thestatic detection field of the detector. Taking, for example, a vesselhaving an interior surface to which an array of optically labeledanalytes is attached, translation of the vessel relative to an opticaldetector can provide an image of the array that is larger than the fieldof view of the detector. Similarly, scanning-based delivery of energycan allow sequential reactions to be carried out in a vessel.

A difficulty that plagues many scanning detectors is that mechanisms fortranslating the vessel relative to the detector are coupled withmechanisms for adjusting rotational registration of the vessel withrespect to the detector. As such, the scanning detector is burdened witha tolerance stack that includes not only translational tolerances butalso rotational tolerances. Relatively small amounts of rolling rotationor pitching rotation (i.e. rotation around the x axis and rotationaround they axis, respectively, as diagrammed in FIG. 1) can havesignificant adverse impacts on high resolution imaging of an analytearray. This adverse impact is exacerbated in optical scanningapplications since a small pitch deviation (i.e. rotation around theyaxis) will manifest as an increasing drift out of focus as the opticaldetector scans a vessel along the x dimension. The longer the scan, thefurther the deviation from focus.

A common solution to the problem of high tolerance stacks in opticalscanners has been to employ moving stages having high precisionactuators that are adjustable in a variety of translational androtational directions. High precision actuators add cost and complexityto a scanner, and such rigs typically require highly trained techniciansfor routine maintenance. Particular embodiments of the apparatus andmethods set forth herein avoid these problems by decoupling themechanism that is used to translate a vessel with respect to a detectorfrom the mechanism that is used to rotationally register the vessel withrespect to the detector. Decoupling translation from rotationalregistration reduces the tolerance stack for the translation mechanismin detection apparatus and other apparatus of the present disclosure.

A further advantage of replacing a typical stage with a vesseltranslation apparatus of the present disclosure is that the vessel canbe scanned more quickly. The increase in scanning speed is, in largepart, a function of the vessel translation apparatus being configured tomove a mass that is smaller than a typical stage. A small mass takesless time to settle compared to a larger mass that is moved the samedistance. For example, the time spent waiting for a vessel to settleprior to acquiring an image becomes increasingly significant as thedesired resolution for detection increases because the motion of thevessel must dampen to a point that the average displacement experiencedby features of the object under observation is small enough to precludesubstantial distortions in the image. Taking as an example a typicalnucleic acid sequencing apparatus, DNA is present in sites of an arraythat are only a few microns apart and that are observed at low micronresolution. A typical stage used to move the array for sequencingrequires settle times of several hundred milliseconds to dampen to thepoint that displacements are less than a few microns. Avoiding a typicalstage by using an apparatus of the present disclosure allows settletimes on the order of a few tens of milliseconds. The milliseconds canadd up to hours for a nucleic sequencing protocol or other repetitivescanning operation. For example, saving 500 hundred milliseconds perimage adds up to a savings of about 4 hours in settling time alone for asequencing protocol that acquires 200 images per cycle and performs 150cycles per run. Similar improvements in processing speed can be achievedfor other scanning applications such as photochemistry,photolithography, microfabrication or nanofabrication (e.g. via laseretching), laser ablation or the like.

Although apparatus and methods set forth herein provide advantages inreducing settle time, it will be understood that the uses need not belimited to processes that include a settling step. Accordingly,apparatus and methods set forth herein in the context of so called “stepand shoot” scanning procedures can be applied to continuous scanningoperations such as time delayed integration (TDI) scanning. For example,apparatus and methods set forth herein can be modified for use in TDIline scanning operations such as those set forth in U.S. Pat. No.7,329,860, which is incorporated herein by reference.

As set forth in further detail herein, rotational registration of avessel with respect to a detector can be achieved by physicallycontacting the vessel with a reference surface, the reference surfacebeing rotationally fixed with respect to the detector. In particularembodiments, as exemplified below, a vessel can be compressed to thereference surface by a preload. Separately, translation can be achievedby a scan actuator (e.g. a gear) that interacts directly with anothersurface of the vessel (e.g. a rail that complements the gear). Thepreload and scan actuator need not interact to achieve motion andregistration of the vessel. For example, the preload need not be appliedto the vessel while the vessel is being translated. However, interactionbetween the preload and scan actuator can occur for certain applicationsof the apparatus and methods set forth herein. Accordingly, the preloadcan be applied to the vessel while the vessel is being translated.

In some embodiments, a vessel that is to be detected can be a componentof a cartridge. The cartridge can provide a convenient mechanism todeliver the vessel to a detector. For example, a detector can bemaintained inside of an analytical instrument to protect the detectorfrom environmental factors such as moisture, dust or light. A cartridgecan be introduced to the analytical instrument via a door or openingsuch that the vessel is contacted with the detector. In someembodiments, the analytical instrument will remove the vessel from thecartridge and translate the vessel past the detector in a way that doesnot necessarily involve movement of the cartridge. Alternatively, thevessel can maintain contact with the cartridge such that both thecartridge and vessel are moved to achieve translation or scanning. In afurther alternative, the cartridge can be a component of the analyticalinstrument and the vessel can be introduced to the instrument by placingthe vessel into the cartridge.

Alternatively and/or additionally, the vessel can be a component of acaddy that also includes reservoirs and fluidic components that deliverreagents to the vessel during the course of a reaction that is detected,such as a nucleic acid sequencing reaction. In some embodiments, thecaddy includes sufficient fluidic components that it functions as a“wet” component and the analytical instrument housing the detectorfunctions as a “dry” component. An advantage of having separate wet anddry components is that the caddy and vessel can be dedicated to aparticular sample or reaction, and when the reaction is complete, thecaddy and vessel can be removed from the analytical instrument andreplaced with a new caddy and vessel dedicated to a second sample orreaction. Because the samples, reagents and reaction products for eachof these two reactions are physically separated from the analyticalinstrument, cross contamination between the reactions, that wouldotherwise cause detection artifacts, are avoided.

The physical separation of the components provides a further advantageof avoiding unnecessary downtime for the analytical instrument if thefluidic component experiences mechanical difficulties. Specifically,unlike many commercially available analytical instruments which havepermanently integrated fluidics, a fluidic system failure can beconveniently overcome by merely removing a faulty fluidic caddy andreplacing it with another so that the analytical instrument experienceslittle to no downtime. In some embodiments, the caddy is disposable, forexample, being made from relatively inexpensive components. The caddycan be configured in a way that reagents are sealed in the caddy therebyavoiding unwanted contamination of the environment and unwanted exposureof laboratory personnel and equipment to the reagents. Alternatively,the fluidics caddy can be emptied, refilled and re-used if desired for aparticular application.

In some embodiments, a fluidic caddy of the present disclosure includesnot only reagent reservoirs, but also includes one or more wastereservoirs. Reagent that is not consumed in a reaction and/or unwantedproducts of a reaction can be collected in the waste reservoir.Advantages of retaining pre- and post-reaction fluids in a caddy includeconvenience of the user in handling a single fluidic component beforeand after a reaction is performed, minimizing user contact with chemicalreagents, providing a compact footprint for the apparatus and avoidingunnecessary proliferation of fluid containers.

Exemplary fluidic caddies, reaction vessels and fluidic components thatcan be modified, in accordance with teachings herein, for use incombination with detection components of the present disclosure aredescribed in commonly owned U.S. patent application Ser. No. 15/922,661,which claims the benefit of U.S. Provisional App. No. 62/481,289, eachof which is incorporated herein by reference. Other fluidic componentsthat are useful, particularly for cyclic reactions such as nucleic acidsequencing reactions, are set forth in US Pat. App. Pub. Nos.2009/0026082 A1; 2009/0127589 A1; 2010/0111768 A1; 2010/0137143 A1; or2010/0282617 A1; or U.S. Pat. Nos. 7,329,860; 8,951,781 or 9,193,996,each of which is incorporated herein by reference.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. The drawings and description areprovided as examples for purposes of explanation and are not necessarilyintended to limit the scope of the invention. The invention issusceptible to modifications in the methods and materials, as well asalterations in the fabrication methods and equipment. Such modificationswill become apparent to those skilled in the art from a consideration ofthe drawings and the description below.

The present disclosure provides a detection apparatus. The apparatus caninclude (a) a vessel having a lumen and a wall, wherein the wall has aninternal surface and an external surface, wherein the internal surfacecontacts the lumen; (b) a reference surface that forms a structural loopwith a detector; (c) a preload configured to urge the external surfaceof the vessel to contact an area on the reference surface; (d) a scanactuator configured to slide the vessel along the reference surface in ascan dimension; and (e) a transmitter configured to direct, to thedetector, a signal from the internal surface or the lumen, when theexternal surface of the vessel is urged by the preload to contact thereference surface.

In particular embodiments, a detection apparatus can include (a) avessel having a lumen and a wall, wherein the wall has an internalsurface and an external surface, wherein the internal surface contactsthe lumen, and wherein the external surface has length l in a scandimension x; (b) a reference surface; (c) a preload configured to urgethe external surface of the vessel to contact an area on the referencesurface, optionally the area of contact can have a maximum length in thescan dimension x that is shorter than length l; (d) a scan actuatorconfigured to slide the vessel along the reference surface in the scandimension x; (e) a detector; and (f) an objective configured to directradiation from the vessel to the detector when the external surface ofthe vessel is urged by the preload to contact the reference surface.

The present disclosure also provides is a method of scanning a vessel.The method can include (a) translating a vessel along a referencesurface of a detection apparatus, wherein the vessel comprises a lumenand a wall, wherein the lumen comprises analytes, wherein the referencesurface contacts at least a portion of the vessel during thetranslating, and wherein the reference surface forms a structural loopwith a detector; and (b) detecting the analytes at different locationsalong the vessel using the detector, wherein the vessel is urged to thereference surface by a preload during the detecting, thereby scanningthe vessel.

In some embodiments, a method of scanning a vessel can include (a)examining a first subset of analytes in a vessel while applying apreload to a first portion of the vessel, wherein the preload positionsthe first subset of analytes to occupy an xy plane in a detection zone,wherein the preload is not applied to a second portion of the vessel;(b) translating the vessel to position a second subset of the analytesin the xy plane of the detection zone; and (c) examining the secondsubset of the analytes in the vessel while applying the preload to asecond portion of the vessel, wherein the preload positions the secondsubset of the analytes to occupy the xy plane of the detection zone,wherein the preload is not applied to the first portion of the vessel,thereby scanning the vessel.

Also provided is a method of optically scanning a vessel. The method caninclude (a) providing a vessel having a lumen and a wall, wherein thelumen contains optically detectable analytes and wherein the wall istransparent to the optically detectable analytes; (b) translating alength of the vessel along a reference surface and detecting theoptically detectable analytes at different locations along the length,wherein the reference surface contacts only a portion of the length ofthe vessel at any time during the translation, wherein the vessel isurged to the reference surface by a preload during the detection,wherein the detection includes transmitting radiation through the wall,then through an objective and then to a detector, thereby opticallyscanning the vessel.

FIG. 2 shows an exemplary arrangement for scanning a vessel relative toa detector. As shown in the profile views of FIG. 2A and FIG. 2B, thevessel is a flow cell 101 that is aligned with objective 110 via a rigidbody 100. The back side of rigid body 100 has a conical depression 116that complements the shape of objective 110. Accordingly, objective 110can be moved close to the flow cell for a desired focus or resolution.Any of a variety of depression shapes can be used as desired toaccommodate the shapes for various objectives or other opticalcomponents. The front side of rigid body 100 has a reference surface 117that will contact a planar face of flow cell 101. The flow cell 101 ismaintained in contact with the reference surface 117 by a preload thatapplies positive pressure to the side of flow cell 101 that is oppositethe reference surface 117. The preload is formed by compression foot 102which contacts flow cell 101 under force of spring 103.

Generally, reference surface 117 and compression foot 102 create lowfriction contacts with flow cell 101. This allows the flow cell to slidepast the reference surface 117 and to slide past compression foot 102while under compression force of the preload. This compression providesalignment of the flow cell 101 with the objective 110 via the rigid bodythroughout the course of flow cell 101 scanning by the objective 110.The reference surface and objective are components of a structural loop.The structural loop contains structural elements that locate the vessel(e.g. flow cell) with respect to the detector (e.g. via the objective).Because the reference surface is pre-aligned with the objective,compressing the flow cell to the reference surface prevents unwantedpitch and roll of the flow cell with respect to the objective.Components of FIG. 2 that are in the structural loop include referencesurface 117, which is connected to rigid body 100, which is connected tobase 114. Base 114 can be connected to a plate or other structuralelement that is physically connected to components of an optical systemsuch as those exemplified in FIG. 9.

In the example shown in FIG. 2, reference surface 117 is polishedaluminum, which provides rigidity for aligning the flow cell 101 to theobjective 110 and a low friction surface for sliding the glass surfaceof the flow cell 101. Any of a variety of materials can be used thatprovide rigidity and low friction for the reference surface including,for example, acetal resins (e.g. Delrin® available from DuPont,Wilmington, Del.), diamond like carbon or polished metals. Thecompression foot 102 provides a low friction surface for the slidingtranslation of the flow cell 101 glass surface and also providescompressibility to form a compliant contact with the flow cell 101 underthe force of spring 103. Any of a variety of materials can be used thatprovide low friction to the compression foot including, for example,those set forth above for reference surface 117. Optionally, a lowfriction material used in an apparatus herein can also be compressible,examples of which include, but are not limited to,polytetrafluoroethylene (PTFE, Teflon®), perfluoroalkoxy alkane (PFA),fluorinated ethylene propylene (FEP), silicone foam, nitrile rubber,Buna-N, Perbunan, acrylonitrile butadiene rubber or nitrile butadienerubber (NBR). Alternatively or additionally, low friction can beachieved using ball bearings, rollers and/or lubricating fluids.Typically, the lubricating fluid is used on the side of the flow cellthat is not between the analytes and detector or a fluid is used thatdoes not interfere with detection. In some embodiments, lubricatingfluids are not present at the interface between the reference surfaceand the exterior surface of the vessel wall. For example, lubricatingfluids can be avoided to prevent interference caused when the fluidenters the area between the detector and vessel.

In particular embodiments, a vessel (or cartridge containing a vessel)is positioned in an xy plane without contacting a reference surface. Forexample, a vessel (or cartridge) can be urged, by a preload, toward afluid bearing or magnetic bearing such that the combination of forcesprovided by the preload and bearing results in a desired positioning. Afluid bearing can be a gas bearing, whereby gas pressure provides aforce for positioning the vessel (or cartridge). Another useful type offluid bearing is a liquid bearing, whereby liquid pressure provides aforce for positioning the vessel (or cartridge). The liquid can beselected for the ability to index match with optical components of thesystem, such as the wall of the vessel, so as to minimize aberrationswhen detecting optical signals or delivering radiation.

As shown in FIG. 2C and FIG. 2D, reference surface 117 has a planarsurface that forms a flat ring on the front face of rigid body 100. Thering is raised compared to the front face of rigid body 100. Raising thereference surface helps to prevent unwanted contact between the flowcell 101 and rigid body 100 that may otherwise create friction thathinders translation. Raising the reference surface 117 also isolates thearea of the flow cell that is to be detected and prevents unwantedwarping that could otherwise occur if the flow cell contacted otherregions of rigid body 101. In the example of FIG. 2, the referencesurface has an area that is smaller than the surface of the flow celland thus only contacts a portion of the flow cell surface. However, inalternative embodiments, the reference surface can be substantially thesame size or larger than the flow cell surface and thus can contactsubstantially all of the flow cell surface (optionally, excepting thearea of the flow cell surface that is juxtaposed with a detectionwindow, objective or other transmitter).

In the example shown, reference surface 117 surrounds circular window118, this window being a hole through rigid body 100. Alternatively,circular window 118 can include a material that is capable oftransmitting a signal that is to be detected. For example, the windowcan be made of quartz, glass, or plastic that facilitates transmissionof signals that are to be detected. In some configurations, the windowcan contain an index matched immersion fluid that contacts the flow cellsurface to facilitate detection, as set forth in further detail belowwith regard to FIG. 11. The circular window 118 is aligned with thefront lens 115 of the objective 110 such that the objective 110 canobserve flow cell 101 through the window 118. Compression foot 102 has aflat ring shape providing a footprint on flow cell 101 that iscomplementary to the footprint of flat ring 117 on the opposite side ofthe flow cell. In this example, the preload (via foot 102) has a contactarea with the vessel (flow cell 101) that is the same as the area ofcontact between reference surface 117 and the vessel. Alternatively, thepreload can have a contact area with the vessel that is smaller than thearea of contact between the reference surface and the vessel. Indeed,the preload can have a contact area with the vessel that is no largerthan the area of contact between the reference surface and the vessel.

Generally, complementarity between the footprints of the preload andreference surface can be configured to result in the compression foot102 having a contact area on the flow cell 101 that excludes surfacearea of the flow cell opposite the circular window 118 and that furtherexcludes surface area of the flow cell opposite the region of the rigidbody that surrounds reference surface 117. Complementarity between thefootprints of compression foot 102 and reference surface 117 helps tomaintain flatness for the portion of the flow cell surface that isobserved through window 118. This complementarity can be beneficial fordetecting analytes on the inner surface of the flow cell, especially athigh magnification and high resolution. The complementarity can alsofacilitate trans-illumination, whereby radiation can pass back or forththrough a path defined by the hollow space in the spring 103,compression foot 102 and window 118. The circular shape of the referencesurface and preload is exemplary. Other shapes can be used including,but not limited to, square, rectangular, polyhedral, elliptical,triangular or the like. Moreover, the shape need not be continuous.Instead the reference surface and/or contact surface for the preload canbe a discontinuous area such as that formed by two parallel tracks or byinterruptions to the above shapes. Particularly useful applications arenucleic acid microarray detection and nucleic acid sequencing. Theshapes and orientations for preload and reference surface can be usedfor apparatus that deliver energy to a vessel or that detect non-opticalsignals.

As exemplified by FIG. 2, a particularly useful vessel for use in adetection apparatus or other apparatus of the present disclosure is aflow cell. Any of a variety of flow cells can be used including, forexample, those that include at least one channel and openings at eitherend of the channel. The openings can be connected to fluidic componentsto allow reagents to flow through the channel. The flow cell isgenerally configured to allow detection of analytes within the channel,for example, in the lumen of the channel or on the inner surface of awall that forms the channel. In some embodiments, the flow cell caninclude a plurality of channels each having openings at their ends. Forexample, the flow cell shown in FIG. 2 has three channels 120, 121 and122 each having openings at both ends. Multiple channels can interactwith a fluidic system via a manifold.

In particular embodiments, a flow cell will include a solid support towhich one or more target analytes or reagents are attached. Aparticularly useful solid support is one having an array of sites.Arrays provide the advantage of facilitating multiplex detection. Forexample, different reagents or analytes (e.g. cells, nucleic acids,proteins, candidate small molecule therapeutics etc.) can be attached toan array via linkage of each different analyte to a particular site ofthe array. Exemplary array substrates that can be useful include,without limitation, a BeadChip™ Array available from Illumina, Inc. (SanDiego, Calif.) or arrays such as those described in U.S. Pat. Nos.6,266,459; 6,355,431; 6,770,441; 6,859,570; or 7,622,294; or PCTPublication No. WO 00/63437, each of which is incorporated herein byreference. Further examples of commercially available array substratesthat can be used include, for example, an Affymetrix GeneChip™ array. Aspotted array substrate can also be used according to some embodiments.An exemplary spotted array is a CodeLink™ Array available from AmershamBiosciences. Another array that is useful is one that is manufacturedusing inkjet printing methods such as SurePrint™ Technology availablefrom Agilent Technologies.

Other useful array substrates include those that are used in nucleicacid sequencing applications. For example, arrays that are used tocreate attached amplicons of genomic fragments (often referred to asclusters) can be particularly useful. Examples of substrates that can bemodified for use herein include those described in Bentley et al.,Nature 456:53-59 (2008), PCT Pub. Nos. WO 91/06678; WO 04/018497 or WO07/123744; U.S. Pat. Nos. 7,057,026; 7,211,414; 7,315,019; 7,329,492 or7,405,281; or U.S. Pat. App. Pub. No. 2008/0108082, each of which isincorporated herein by reference.

An array can have sites that are separated by less than 100 μm, 50 μm,10 μm, 5 μm, 1 μm, or 0.5 μm. In particular embodiments, sites of anarray can each have an area that is larger than about 100 nm², 250 nm²,500 nm², 1 μm², 2.5 μm², 5 μm², 10 μm², 100 μm², or 500 μm².Alternatively or additionally, sites of an array can each have an areathat is smaller than about 1 mm², 500 μm², 100 μm², 25 μm², 10 μm², 5μm², 1 μm², 500 nm², or 100 nm². Indeed, a site can have a size that isin a range between an upper and lower limit selected from thoseexemplified above. An array can have sites at any of a variety ofdensities including, for example, at least about 10 sites/cm², 100sites/cm², 500 sites/cm², 1,000 sites/cm², 5,000 sites/cm², 10,000sites/cm², 50,000 sites/cm², 100,000 sites/cm², 1,000,000 sites/cm²,5,000,000 sites/cm², or higher. An embodiment of the apparatus ormethods set forth herein can be used to image an array at a resolutionsufficient to distinguish sites at the above densities or siteseparations.

Several embodiments utilize optical detection of analytes in a flowcell. Accordingly, a flow cell can include one or more channels eachhaving at least one transparent window. In particular embodiments, thewindow can be transparent to radiation in a particular spectral rangeincluding, but not limited to x-ray, ultraviolet (UV), visible (VIS),infrared (IR), microwave and/or radio wave radiation. In some cases,analytes are attached to an inner surface of the window(s).Alternatively or additionally, one or more windows can provide a view toan internal substrate to which analytes are attached. Exemplary flowcells and physical features of flow cells that can be useful in a methodor apparatus set forth herein are described, for example, in US Pat.App. Pub. No. 2010/0111768 A1, WO 05/065814 or US Pat. App. Pub. No.2012/0270305 A1, each of which is incorporated herein by reference inits entirety.

Several examples herein are demonstrated for a rectangular flow cell 101having elongated channels. In these examples, the area of contactbetween the flow cell 101 and reference surface 117 has a maximum lengthin the scan dimension x that is shorter than the length of the flow celllane in scan dimension x. More specifically, the diameter of ring 117 isshorter than the length of lanes 120, 121 or 122. Alternatively oradditionally, the area of contact between the flow cell 101 andreference surface 117 can have a maximum width w in dimension y that isshorter than the width of the flow cell lane in dimension y.Specifically, the diameter of ring 117 can be shorter than the width ofany one of lanes 120, 121 or 122.

Similarly, the maximum diameter or length of window 118 in the scandimension x can be shorter than the length of the flow cell lane in thescan dimension x. Alternatively or additionally, the maximum diameter orwidth of window 118 in the y dimension can be shorter than the width ofany one of lanes 120, 121 or 122. In this configuration, the completewidth of the lane can be observed by translation in they direction. Insome embodiments, the area of window 118 and width of the lane can beconfigured so that translation in they dimension is not necessary toobserve the entire width of the lane. For example, the area of window118 can have a maximum diameter or width w in dimension y that isequivalent to or longer than the width of the flow cell lane indimension y.

In particular embodiments a vessel, such as a flow cell, can be moved inan arcuate path during all or part of a scanning operation. Looking tothe flow cell orientation in FIG. 1, the arcuate path can result fromrotation around the yaw axis. The arcuate path can be a circle, spiralor other path that is desirable for scanning a vessel. Optionally, thearea of contact between a vessel and reference surface can have a lengthor area that is smaller than the length or area, respectively, of thearcuate path. By way of more specific example, a ring-shaped referencesurface can have a diameter that is shorter than the length of thearcuate path or shorter than the length of a lane in a flow cell that ismoved along the arcuate path. Similarly, the maximum diameter or area ofa window in the reference surface, through which detection occurs, canbe smaller than the length or area, respectively, of the arcuate path;or the window can be smaller than a flow cell lane that is scanned alongan arcuate path.

A flow cell need not be rectangular in shape. Alternative shapes thatcan be used include, but are not limited to, a disc, square, polygon orirregular shape. The lanes of a flow cell can follow a linear path,arcuate path, winding path or the like. Other types of vessels can alsobe used. For example, a well of a multi-well strip or multi-well platecan be detected using an apparatus or method of the present disclosure.The bottom surface of a well can be urged toward a reference surface bya preload applied to the top of the vessel (e.g. by contacting acompression foot to the upper side of a multi-well plate or multi-wellstrip). Optionally, the well can have a flat bottom that contacts thereference surface. As a further option, the well will be larger than thefield of view of the detector. For example, the well may be circular inshape and may have a diameter € in scan dimension x that is longer thanthe length of the reference surface in the scan dimension x.

Another exemplary vessel type is a cylindrical- or tube-shaped vesselsuch as a capillary tube. The body of a tube can be held to a referencesurface under the force of a preload as exemplified herein for flatshaped vessels. In an exemplary configuration the length of the tube canbe parallel to the scan axis such that scanning the tube along x willresult in relative motion of the reference surface along the length ofthe tube. For a tube that is configured in this orientation, it may alsobe useful to rotate the tube in the roll axis. This rotation will resultin relative motion of the reference surface around the circumference ofa section of the tube. Combining translation along x and rotation alongthe roll axis can allow a substantial surface area of the tube to comeinto contact with the reference surface. For example, the tube andreference surface can move in a helical or spiral path relative to eachother. The reference surface can be flat, as exemplified herein for flowcells having a flat exterior wall. Alternatively, the reference surfacecan have a curved shape (e.g. u-shaped or saddle-shaped cross section)that accommodates and orients a cylindrical- or tube-shaped vessel thatit contacts.

Typically, the vessel wall is made from a rigid material that is notreadily flexible under the conditions used. In alternative embodiments,a vessel is made from a flexible material, for example, forming a sheet,tape, belt or ribbon that can be passed along a reference surface anddetected while the vessel is under the urging of a preload. For example,a plurality of analytes, such as an array of nucleic acids, can beattached to the surface of the flexible material and detected when incontact with the reference surface. Exemplary, flexible materials havingattached analytes are described, for example, in U.S. Pat. No. 9,073,033and US Pat. App. Pub. No. 2016/0076025 A1, each of which is incorporatedherein by reference.

When using a vessel having a flexible wall, it may be advantageous topull the wall material over a reference surface, for example, to stretchor straighten the portion of the wall material that is observed by adetector. For example, the reference surface can be a raised rim thatsurrounds a detection window and the flexible material can be pulledover the rim to apply a pulling force across the window. Pulling can beachieved for example by applying suction to the flexible material via avacuum chuck that surrounds the raised rim. Suction can be applied as analternative or supplement to other preload mechanisms set forth herein.

As will be evident from the examples set forth herein, a vessel can beopen (e.g. a well of a multi-well plate, surface of a chip, or surfaceof a sheet) or the vessel can be enclosed (e.g. a lane of a flow cell).It will be understood that, wells of a multi-well plate can optionallybe covered to create an enclosed vessel and similarly a sheet, belt,tape or ribbon can have multiple layers such that an internal lumenoccurs between layers. Alternatively, a vessel can have one or more openstructures such as a trough, well or other concave structure thatcontains a fluid. A vessel can also have a convex or protrudingstructure such as a post or ridge, and optionally individual protrusionscan each be attached to one or more analyte that is to be detected ormanipulated.

The preload exemplified in FIG. 2 creates a pushing force on the side ofthe vessel (e.g. flow cell) that is opposite the side of the vessel thatcontacts the reference surface. Pushing force can derive from a spring,clamp, positive air pressure, positive fluid pressure, charge repulsion,charge attraction, magnetic attraction or magnetic repulsion.Alternatively, a preload can be configured to create a pulling force onthe vessel. For example, a magnetic or ferromagnetic material that is inor on the vessel can be attracted to the reference surface, or chargesin or on the vessel can be attracted to the reference surface. In thisexample, the reference surface or area surrounding the reference surfacecan contain magnetic or ferromagnetic material that acts as a preload.In another embodiment, pulling force can result from a vacuum chuck thatis configured to apply suction to an area of the vessel that contactsthe reference surface. In a further embodiment, a magnetic clampingforce can be used, whereby the vessel is sandwiched between a magneticor ferromagnetic material on or around the reference surface thatattracts a magnetic or ferromagnetic body that is external to theopposite side of the vessel.

A detection apparatus or other apparatus of the present disclosure caninclude a scan actuator that is configured to slide a vessel along areference surface. The vessel can slide along the reference surface andalong the surface of the preload. Generally, the scan actuator isconfigured to move the vessel while the vessel is in contact with thereference surface under the urging of a preload. However, it is alsopossible to translate the vessel without simultaneously applying apreload to the vessel. It is also possible to translate the vesselthrough a space defined by a bearing that does not physically contactthe vessel, such as a fluid bearing or magnetic bearing. For example, avessel can be positioned via opposing forces of a preload against abearing. Particularly useful actuators employ one or more gears thatinteract with perforations or threads on a flow cell or on a cartridgethat contains the flow cell. Several examples are set forth below.

In some embodiments, the scan actuator can use a film sprocketmechanism. The vessel that is to be translated, or a cartridge thatholds the vessel, can contain a track of perforations that engages asprocket in a detection apparatus to achieve translation. As shown inthe exemplary configuration of FIG. 3, flow cell 101 is housed incartridge 125, which contains two perforation tracks 130 and 140.Perforation track 130 is located near the top edge of the cartridge 125and runs parallel to the longest dimension € of the flow cell.Perforation track 140 is located near the opposite edge of the cartridge125 and also runs parallel to €. Sprockets 150 and 160 are configured toengage perforation tracks 130 and 140, respectively, when urged towardreference surface 117 by the force of preload spring 103. The flow cell101 can be translated in scan dimension x, which is parallel to f, byrotating the engaged sprockets 150 and 160.

FIG. 4A shows a cartridge 400 having an inset 403 for flow cell 430. Theinset includes notches 404 and 405 that are placed to facilitateadjustment or removal of the flow cell 430. Cartridge 400 has a singleperforation track 401 near the top edge 402. As shown in FIG. 4B, theperforations are complementary to teeth on sprocket 420 and perforationtrack 401 is inset into the face of cartridge 400 thereby providing atrack that engages guide 410. Guide 410 slots into perforation track 401to prevent rotation of cartridge 400 in the yaw axis during translationunder the action of sprocket 420, thereby preventing unwanted yawrotation of the flow cell 430 relative to a detector. As shown in FIG.4C, flow cell 430 includes a bottom plate 431 that is sized for pressurefit with inset 403 and also includes a top plate 440. A channel 443 isformed between plates 431 and 440 due to presence of a spacer or gasket.The top plate 440 also includes holes 441 and 442 which act as inlet andoutlet for channel 443. A perspective view of the cartridge 400 withassembled flow cell 430, sprocket 420 with motor 425, and guide 410 isshown in FIG. 4D.

Another useful mechanism for scan actuation is a spur gear that engagesteeth on an edge of a flow cell, or on an edge of a cartridge holdingthe flow cell. FIG. 5A shows cartridge 200 which is pressure fitted toflow cell 101, and which has a serrated bottom edge 240 and smooth topedge 241. Serrated bottom edge 240 engages spur gear 230 when cartridge200 is urged by preload spring 103 to contact a reference surface onrigid body 100. The cartridge 200 and flow cell 101 are translated byrotating spur gear 230. Wheel guides 210 and 220 engage the smooth edge241 of the cartridge 200, when the cartridge 200 is positioned tocontact the flow cell 101 with a reference surface on rigid body 100.The wheel guides function to prevent rotation of the cartridge 200 andflow cell 101 about the yaw axis.

Scan actuation can also employ a ball screw that engages a threadedcatch on a flow cell, or on a cartridge holding the flow cell. FIG. 6Ashows cartridge 300 which is pressure fitted to flow cell 101, and whichhas a threaded catch 311 on the top and two guide catches 312 and 313 onthe bottom. Threaded catch 311 engages screw 310 when cartridge 300 isurged by preload spring 103 to contact a reference surface on rigid body100. The cartridge 300 and flow cell 101 are translated by rotatingscrew 310 against threads of catch 311. Guide catches 312 and 313 engagerail 320, when the cartridge 300 is positioned to contact the flow cell101 with reference surface 117. The guide catches 312 and 313 functionto prevent rotation of the cartridge 300 and flow cell 101 about the yawaxis.

Scan actuation can use mechanical contact between the motor and vessel(or vessel cartridge) as exemplified above. Alternatively oradditionally, interaction between motor and vessel (or vessel cartridge)can be mediated by magnetic attraction. For example, the vessel orcartridge can have a magnetic or ferromagnetic material that interactswith a magnetic or ferromagnetic component of the actuator.

Whether using mechanical contact or other interactions to mediateactuation, a linear motor can be used to drive the scanning motion.Exemplary linear motors that can be used include synchronous linearmotors, induction linear motors, homopolar linear motors and piezoelectric linear motors.

An apparatus of the present disclosure can further include a y actuatorconfigured to change the relative translational position of the detectorand the vessel along they dimension. Taking as an example the apparatusshown in FIG. 2, a y actuator can operate, for example, by changing therelative translational position of the objective 110 and the referencesurface 117. Alternatively or additionally, a y actuator can operate bychanging the relative translational position of the flow cell 101 andthe reference surface 117. Translation along they dimension can allowdifferent lanes of a flow cell to be addressed. When a lane is widerthan the field of view for the objective, y translation can be used todetect multiple swaths of the lane (i.e. a first swath can be detectedby a scan along x and a second swath can be addressed by a step alongthe y dimension followed by a second scan along x). A y actuator can beconfigured similarly to the x actuators exemplified herein. For example,a y actuator can be configured to translate the flow cell while it isurged to a reference surface by a preload. Other stepper motors ortranslation actuators can be used as well for x or y translation.

In particular embodiments, an apparatus of the present disclosure caninclude a rotational actuator configured to change the relativetranslational position of the detector and the vessel along an arcuatepath. Taking the exemplary flow cell oriented as shown in FIG. 1 arotational actuator can rotate the flow cell in the yaw axis. Rotationin the yaw axis can be particularly useful for scanning lanes orfeatures that follow an arcuate path. An additional or alternativerotational actuator can rotate a vessel along the roll axis. Rotation inthe yaw axis can be particularly useful when the vessel is a tube orcylinder that is oriented to have its length along the x axis.

Several embodiments of the present disclosure are exemplified withregard to an objective having several lenses for gathering and focusingradiation from an object (e.g. a vessel such as a flow cell). It will beunderstood that any of a variety of optical elements can serve as anobjective in an apparatus or method of the present disclosure including,for example, a lens, mirror, fiber optic, fiber bundle, lens array orother optical element that gathers radiation from an object beingobserved, whether or not the optical element is also capable of focusingthe radiation. Objectives or other optical components used in anapparatus or method set forth herein can be configured to transmitradiation in any of a variety of spectral ranges including, but notlimited to X-ray, ultraviolet (UV), visible (VIS), infrared (IR),microwave and/or radio wave ranges.

An objective that is used in an apparatus set forth herein can be placedto direct radiation from the internal surface or the lumen of a vessel,through the wall of the vessel and to a detector when the externalsurface of the vessel contacts a reference surface. In particularembodiments, an objective, and other optional components of an opticalsystem, can be configured for epi-illumination luminescence detection(i.e. epi-luminescence), whereby excitation radiation is directed from aradiation source, through the objective, then through the wall of thevessel to the internal surface or the lumen of the vessel; and wherebyemission from the internal surface or the lumen of the vessel isdirected back through the wall and through the objective (i.e.excitation and emission both pass through the objective). Alternatively,objectives, and other optional components of an optical system, can beconfigured for trans-illumination fluorescence, whereby excitationradiation is directed from a radiation source through a first wall of avessel to the internal surface or the lumen of the vessel; and wherebyemission from the internal surface or the lumen of the vessel isdirected through another wall of the vessel and through the objective(i.e. emission passes through the objective, excitation does not). Otheruseful configurations for fluorescence detection include those thatexcite a vessel via total internal reflection fluorescence (TIRF) or viawaveguides. In any of a variety of configurations, the radiation sourcecan form a structural loop with a reference surface such that a vesselthat contacts the reference under the urging of a preload will beproperly oriented with respect to the radiation source.

The objectives shown in FIGS. 2, 3, 5 and 6 are exemplary, having 4lenses. Any number or type of lenses can be included to suit aparticular application. Particularly useful objectives will have anumerical aperture that is at least 0.1 and at most 0.9. Numericalapertures above 0.95 can be achieved using an immersion objective as setforth in further detail below. An objective or other transmitter can beconfigured to operate with a detection system that resolves features(e.g. nucleic acid sites) on a surface that are separated by less than100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. The detection system,including objective or other transmitter, can be configured to resolvefeatures having an area on a surface that is smaller than about 1 mm²,500 μm², 100 μm², 25 μm², 10 μm², 5 μm², 1 μm², 500 nm², or 100 nm².

An optical system used in an apparatus or method set forth herein canhave a field of view that is at least 0.1 mm², 0.5 mm², 1 mm², 2 mm², 3mm², 4 mm² or higher. Alternatively and/or additionally, the field ofview can be configured to be at most 4 mm², 3 mm², 2 mm², 1 mm², 0.5mm², 0.1 mm², or less.

The objective, or other appropriate component of a detection system usedin an apparatus set forth herein, can be configured to focus on analytesthat are in or on the vessel. For example, the apparatus can include afocus actuator configured to change the relative position of theobjective and the reference surface in the focus dimension z. Physicallyaligning the vessel to the reference surface under force of a preloadeffectively fixes the position of the vessel in the z dimension, therebyfavoring accurate and robust focusing throughout a scanning operation.

An apparatus set forth herein can employ optical sub-systems orcomponents used in nucleic acid sequencing systems. Several suchdetection apparatus are configured for optical detection, for example,detection of fluorescent signals. Examples of detection apparatus andcomponents thereof that can be used to detect a vessel herein aredescribed, for example, in US Pat. App. Pub. No. 2010/0111768 A1 or U.S.Pat. Nos. 7,329,860; 8,951,781 or 9,193,996, each of which isincorporated herein by reference. Other detection apparatus includethose commercialized for nucleic acid sequencing such as those providedby Illumina™, Inc. (e.g. HiSeg™, MiSeg™, NextSeg™, or NovaSeg™ systems),Life Technologies™ (e.g. ABI PRISM™, or SOLiD™ systems), PacificBiosciences (e.g. systems using SMRT™ Technology such as the Sequel™ orRS II™ systems), or Qiagen (e.g. Genereader™ system). Other usefuldetectors are described in U.S. Pat. Nos. 5,888,737; 6,175,002;5,695,934; 6,140,489; or 5,863,722; or US Pat. Pub. Nos. 2007/007991 A1,2009/0247414 A1, or 2010/0111768; or WO2007/123744, each of which isincorporated herein by reference in its entirety. In particularembodiments, the stage of a known sequencing system can be replaced witha scanning apparatus set forth herein.

Generally, an objective is the optical element of the detectionapparatus that is proximal (i.e. closest to) the vessel that is to bedetected (e.g. flow cell). In some embodiments, the vessel need notinclude any optical components. In alternative embodiments, one or moreoptical component, such as a lens or fiber optic, can be provided by avessel or by a cartridge to which the vessel is attached. For example,the objective of the detection apparatus can be configured to directexcitation, emission or other signals to the optical component that ispresent on the vessel or cartridge. Thus, the optical component that isproximal to the sample can be provided by the detection apparatus, oralternatively, by the vessel that houses the sample.

A detection apparatus that is used to observe a vessel in a method orapparatus set forth herein need not be capable of optical detection. Forexample, the detector can be an electronic detector used for detectionof protons or pyrophosphate (see, for example, US Pat. App. Pub. Nos.2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; or 2010/0282617 A1,each of which is incorporated herein by reference in its entirety, orthe Ion Torrent™ systems commercially available from ThermoFisher,Waltham, Mass.) or as used in detection of nanopores such as thosecommercialized by Oxford Nanopore™, Oxford UK (e.g. MinION™ orPromethION™ systems) or set forth in U.S. Pat. No. 7,001,792; Soni &Meller, Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2, 459-481(2007); or Cockroft, et al. J. Am. Chem. Soc. 130, 818-820 (2008), eachof which is incorporated herein by reference.

In a particular embodiments, apparatus or methods set forth herein canbe configured for scanning electron microscopy (SEM). Accordingly, anelectron beam can be produced by an electron gun and directed to avessel by one or more condenser lenses, scanning coils and/or deflectorplates. Signal can be detected using an electron detector such as ascintillator-photomultiplier system (e.g. an Everhart-Thornleydetector).

In particular embodiments, a detection apparatus or other apparatus ofthe present disclosure can provide temperature control of a vessel thatis to be detected. Temperature control can be provided by controllingtemperature of an internal chamber that houses the vessel. Alternativelyor additionally, a vessel that is to be detected can be placed intocontact with a thermally conductive surface that is temperaturecontrolled. FIG. 7A shows an exemplary configuration for achievingtemperature control of a flow cell via contact with a thermallyconductive surface. The backside of aluminum body 460 is attached to twothermal elements 450 and 451 which are located left and right of conicaldepression 416. The thermal elements can be polyimide thermofoilheaters, Peltier elements, metal heating elements, ceramic heatingelements, polymer PTC heating elements or the like. Aluminum body 460also includes two legs 461 and 462 for attachment to the detectionapparatus. As such the two legs form part of the structural loop betweenthe reference surface on the aluminum body 460 and the detectionapparatus. Optionally, legs 461 and 462 can be made from a materialhaving low thermal conductivity. Thus, the legs can function to attachthe aluminum body to a detection apparatus in a way that insulates othercomponents of the detection apparatus from experiencing unwantedtemperature fluctuations. Thermal elements 450 and 451 can be activatedvia wires 452 and 453 to heat or cool aluminum body 460 such that a flowcell in cartridge 400 is in contact with the opposite side of aluminumbody 460 and thus is temperature controlled. As shown in FIG. 7B,conical depression 416 is configured to accept an objective 410 fordetection of a flow cell in cartridge 400 through window 418. In theconfiguration shown, the flow cell cartridge 400 is translated via filmsprocket 420 under the control of rotary motor 425.

A detection apparatus or other apparatus of the present disclosure caninclude a fluidics system for delivering reagents to a vessel that is tobe detected. Accordingly, one or more reservoirs can be fluidicallyconnected to an inlet valve of the vessel. The apparatus can furtherinclude a pressure supply for driving reagents from reservoirs to thevessel. The apparatus can include a waste reservoir that is fluidicallyconnected to the vessel to remove spent reagents. Taking as an examplean embodiment where the vessel is a flow cell, reagents can be deliveredvia pump to the flow cell through the inlet and then the reagents canflow through the flow cell outlet to a waste reservoir. The reservoirscan include reagents for any of a variety of analytical proceduresincluding, but not limited to nucleic acid sequencing, nucleic acidgenotyping, nucleic acid expression analysis, protein sequencing,protein binding analysis (e.g. ELISA), small molecule receptor binding,protein phosphorylation analysis, nucleic acid synthesis or proteinsynthesis. Alternatively or additionally, the reservoirs can includereagents for a preparative process. Exemplary preparative processesinclude, but are not limited to, nucleic acid synthesis, peptidesynthesis, assembly of oligonucleotides into genes, photolithography,nanofabrication or microfabrication (e.g. via laser etching), laserablation, or the like.

A fluidic system can include at least one manifold and/or at least onevalve for directing reagents from reservoirs to a vessel where detectionoccurs. Manifolds are particularly useful in sequencing instruments dueto the relatively large number of different reagents that are deliveredduring a sequencing protocol. Exemplary protocols and useful reagentsare set forth in further detail below and in references that areincorporated herein by reference. Fluid flow from the reservoirs can beselected via valves such as a solenoid valve (e.g. those made byTakasago Electric, Japan), ball valve, diaphragm valve or rotary valve.

One or more fluidic components used in a detection apparatus or otherapparatus of the present disclosure can be housed in a fluidic caddythat is separable from detection components. An exemplary fluidic caddy600 is shown in FIG. 8A. Fluidic caddy 600 includes a housing 601 havingsufficient internal volume to house reagent reservoirs 603, wastereservoirs 602, and a piston shaft 604 for an external pump. Any of avariety of fluidic components can be housed in a fluidic caddyincluding, but not limited to, one or more reservoirs, fluid lines,valves or pumps. The fluidic caddy includes latches 610 and 611 whichare configured to engage with hooks in a detection apparatus. See forexample, switch hook 701 in FIG. 9. Flow cell 430 is held withincartridge 400 and cartridge 400 is held to the fluidic caddy 600 viahook 616 and guides 616 and 617. As shown in the expanded cutout of FIG.8B and in side-view FIG. 8D, hook 615 includes a tooth 614 that insertsinto track 401 to hold the cartridge 400 in place. Guides 616 and 617complete a three-point attachment by engaging the bottom edge ofcartridge 400. Preload 620, although shown in retracted position in FIG.8D, can be extended to push against the back side of the cartridge 400,thereby functioning with hook 615 and guides 616 and 617 to hold thecartridge in place by compressive forces.

Fluidic caddy 600 includes openings as shown in FIG. 8D and FIG. 8F. Forpurposes of showing fluidic connections for the flow cell 430, FIG. 8Fshows a perspective view of caddy 600 that has been emptied of severalother fluidic components. Opening 605 is configured to accept the pistonof an external pump. The piston can be driven by a detection apparatusto allow control of fluid flow through flow cell 430 during ananalytical procedure (e.g. a nucleic acid sequencing procedure), but thepiston need not directly contact any fluids in the caddy 600 or in theflow cell 430. Accordingly, the detection apparatus can constitute a“dry” component that does not make direct contact with fluids, whereasthe caddy 600 and flow cell 430 constitute “wet” components. Fluidiccaddy 600 includes two elongated openings 621 and 622 which areconfigured to accommodate tubes 661 and 662, respectively. The elongatedshape allows the tubes to move along the x dimension as the flow cell istranslated during scanning. Thus, the tubes can remain engaged with theflow cell and fluidic reservoirs during a scanning operation.

The flow cell 430 can be translated independently of caddy 600 viamovement of the cartridge as set forth previously herein, for example,in connection with FIG. 4. As such, caddy 600 remains stationary whileflow cell 430 is moved. Alternatively, a flow cell can be attached to acaddy such that the caddy and flow cell are translated as a unit. In afurther alternative, one or more detection components of a detectionapparatus can be moved while the flow cell and/or fluidic caddy isstationary.

Interactions between fluidic caddy 600 and components of a detectionapparatus are shown in FIG. 9. The perspective view in FIG. 9A and topview in FIG. 9B, show caddy 600 engaged in a way that sandwiches flowcell cartridge 400 between the caddy 600 and aluminum body 460. Whenengaged, the flow cell cartridge 400 contacts film sprocket 420 suchthat motor 425 can drive translation of the flow cell therein.Translation will cause the flow cell to move past objective 721 which isin turn configured to direct fluorescence excitation from fluorometer720 to the flow cell and to direct fluorescence emission from the flowcell to fluorometer 720.

The mechanism of engaging caddy 600 and flow cell cartridge 400 with adetection apparatus or other apparatus of the present disclosure can beakin to inserting an 8-track cassette into an audio player. The flowcell 430 and cartridge 400 are connected to caddy 600 such that a userneed not directly handle the flow cell 430, instead delivering it to thedetection apparatus by handling the caddy 600, much like a user need nothandle the tape inside of the 8-track cassette. Similarly, individualfluidic components need not be individually handled but can properlyengage with actuators in the detection apparatus when the caddy 600 isproperly placed in the detection apparatus.

Fluidic caddy 600 is disengaged from the detection apparatus in FIG. 9C,which illustrates mechanical elements that can be used by the detectionapparatus to control function of the fluidic caddy 600. The detectionapparatus can include a sensor or switch that responds to presence ofthe fluidic caddy and actuates functional interactions. In the exampleof FIG. 9, switch hook 701 is displaced when caddy 600 is properlyengaged. This displacement can activate one or more functions. Forexample, the underside of fluidic caddy 600 can include one or moreopenings that are positioned to accept one or more valve actuator 711 onplatform 710. Valve actuators, although shown in the proud position forpurposes of illustration, can be retracted into platform 710 whenfluidic caddy 600 is not present. The valve actuators can be raised inresponse to displacement of switch hook 701 and/or in response tocontrol software for the detection apparatus. Accordingly, the one ormore valve actuator 711 can be used to control flow of fluids to theflow cell, from the flow cell, and/or between reservoirs within thecaddy. In another example, pump component 702 of the detection apparatuscan engage with fluidic components of the caddy 600 via opening 710, forexample, by inserting a piston. Interaction of pump component 702 withthe fluidic caddy 600 can be actuated directly due to displacement ofswitch hook 701 and/or in response to control software for the detectionapparatus.

The structural loop between the flow cell 430 and fluorometer 720includes reference surface 417, aluminum body 460, legs 461 and 462, aplate or base to which legs 461 and 462 are attached, and fluorometer720 which is also attached to the plate or base.

FIG. 10 shows a mechanism that can be used for engaging a flow cell witha detection apparatus. FIG. 10A shows a side view and expanded detail offluidic cartridge 600 and flow cell cartridge 400 when not engaged witha detection apparatus. When the fluidic caddy 600 is not engaged, flowcell cartridge 400 is in contact with hook 615 and guides 616 and 617.FIG. 10B shows an expanded detail of the configuration that results whencaddy 600 is engaged with the detection apparatus. Specifically, flowcell cartridge 400 is moved toward the wall of caddy 600, disengagingfrom hook 615 and from guides 616 and 617.

A mechanism for changing the position of the flow cell cartridge 400 isshown in FIG. 10E, which is a detail view of the interface between caddy600, flow cell cartridge 400 and aluminum body 460. FIG. 10E is a detailof FIG. 10D which is a cutaway along line m in FIG. 10C. When the caddy600 is properly engaged with the detection apparatus, hook 615 andguides 616 and 617 are inserted into notches 471, 472 and 473 inaluminum body 460. The notches 471, 472 and 473 have a sufficient depththat compression of the caddy toward the aluminum body 460 causes thefront side of flow cell cartridge 400 to engage sprocket 420 and thefront side of flow cell 430 to contact reference surface 417. Thecompression also results in the back side of flow cell cartridge 400contacting compression foot 102. In this way, the flow cell 430 ispressed against the reference surface 417 for alignment with objective410, which observes the flow cell 430 through window 418. The flow cell430 can be translated via interaction of sprocket 420 with perforationtrack 401.

Although interactions between a fluidic caddy and detection apparatushave been exemplified herein using mechanical contacts, it will beunderstood that other mechanical switching mechanisms can be used.Electronic switches can also be used, including for example, those thatare activated by electronic sensors (e.g. Bluetooth), magnetic sensors,radio frequency sensors (e.g. RFID), pressure sensors, optical sensors(e.g. barcodes) or the like.

The fluidic caddy and components set forth above are exemplary. Otherfluidic caddies and fluidic components that can be used with a detectionapparatus of the present disclosure are set forth in commonly owned U.S.patent application Ser. No. 15/922,661, which claims the benefit of U.S.Provisional App. No. 62/481,289, and US Pat. App. Pub. No. 2017/0191125A1, each of which is incorporated herein by reference. Moreover, asimilar fluidic caddy can be used with other apparatus of the presentdisclosure, such as reactor apparatus, and the other apparatus can beconfigured as set forth above to interface with a caddy.

Optionally, a detection apparatus or other apparatus of the presentdisclosure can further include a computer processing unit (CPU) that isconfigured to operate one or more of the system components set forthherein. The same or different CPU can interact with the system toacquire, store and process signals (e.g. signals detected in a methodset forth herein). In particular embodiments, a CPU can be used todetermine, from the signals, the identity of the nucleotide that ispresent at a particular location in a template nucleic acid. In somecases, the CPU will identify a sequence of nucleotides for the templatefrom the signals that are detected.

A useful CPU can include, for example, one or more of a personalcomputer system, server computer system, thin client, thick client,hand-held or laptop device, multiprocessor system, microprocessor-basedsystem, set top box, programmable consumer electronic, network PC,minicomputer system, mainframe computer system, smart phone, ordistributed cloud computing environment that includes any of the abovesystems or devices. The CPU can include one or more processors orprocessing units, a memory architecture that may include RAM andnon-volatile memory. The memory architecture may further includeremovable/non-removable, volatile/non-volatile computer system storagemedia. Further, the memory architecture may include one or more readersfor reading from and writing to a non-removable, non-volatile magneticmedia, such as a hard drive, a magnetic disk drive for reading from andwriting to a removable, non-volatile magnetic disk, and/or an opticaldisk drive for reading from or writing to a removable, non-volatileoptical disk such as a CD-ROM or DVD-ROM. The CPU may also include avariety of computer system readable media. Such media may be anyavailable media that is accessible by a cloud computing environment,such as volatile and non-volatile media, and removable and non-removablemedia.

The memory architecture may include at least one program product havingat least one program module implemented as executable instructions thatare configured to control one or more component of an apparatus setforth herein or to carry out one or more portions of a method set forthherein. For example, executable instructions may include an operatingsystem, one or more application programs, other program modules, andprogram data. Generally, program modules may include routines, programs,objects, components, logic, data structures, and so on, that performparticular tasks such as processing of signals detected in a method setforth herein.

The components of a CPU may be coupled by an internal bus that may beimplemented as one or more of any of several types of bus structures,including a memory bus or memory controller, a peripheral bus, anaccelerated graphics port, and a processor or local bus using any of avariety of bus architectures. By way of example, and not limitation,such architectures include Industry Standard Architecture (ISA) bus,Micro Channel Architecture (MCA) bus, Enhanced ISA (EISA) bus, VideoElectronics Standards Association (VESA) local bus, and PeripheralComponent Interconnects (PCI) bus.

A CPU can optionally communicate with one or more external devices suchas a keyboard, a pointing device (e.g. a mouse), a display, such as agraphical user interface (GUI), or other device that facilitatesinteraction of a user with the nucleic acid detection system. Similarly,the CPU can communicate with other devices (e.g., via network card,modem, etc.). Such communication can occur via I/O interfaces.Furthermore, a CPU of a system herein may communicate with one or morenetworks such as a local area network (LAN), a general wide area network(WAN), and/or a public network (e.g., the Internet) via a suitablenetwork adapter.

FIG. 11 shows a cutaway profile view of an exemplary optical arrangementthat uses immersion optics. The arrangement includes an objective 710that includes a housing 720 and several lenses 711, 712 and 715. Thenumber, position and shape of the lenses is exemplary and can varyaccording to desired prescription. Also included is rigid body 700, flowcell 701 and flow cell cartridge 702. Flow cell cartridge 702 includesinlet 741 and outlet 742 for moving fluid reagents into and out of theflow cell. The bottom side of rigid body 700 has a reference surface 717that becomes sealed by flow cell 710 when a preload is applied, forexample, as set forth using configurations set forth above. Oppositethis seal, rigid body 700 includes a conical depression 716 that isshaped to accept the tip of objective 710. The space 716 between rigidbody 700, objective 710 and the seal can be filled with an immersionfluid, such as an oil or aqueous solvent that is index matched to theobjective. As such, the immersion fluid will directly contact theproximal lens 715 of objective 710 and the surface of flow cell 701. Thefluid can be maintained in the space 716 by seals 731 and 732, which areoptionally flexible. Fluid can be added and/or removed from space 716via line 733. Immersion optics can provide several advantages overoptics that image through air including, for example, the ability toachieve numerical aperture (NA) greater than 0.95, ability to image atgreater depths into a vessel, and alleviating tolerances on thethickness and uniformity of vessel walls through which the objectiveresolves objects.

The present disclosure provides methods that are particularly useful forperforming cyclical reactions. Each cycle can include deliveringreagents for the reaction to a flow cell or other vessel where,optionally, the reaction, or products of the reaction, will be observed.Each cycle can further include scanning of the vessel using apparatus ormethods set forth herein. The methods are exemplified herein in thecontext of a nucleic acid sequencing reaction. However, those skilled inthe art will understand from the teaching herein how to modify themethods, and the apparatus, for other cyclical reactions such as nucleicacid synthesis reactions, peptide sequencing reactions, peptidesynthesis reactions, combinatorial small molecule synthesis reactions orthe like. However, the method need not be cyclical and can instead becarried out in a non-repetitive configuration, for example, to observe asingle reaction or phenomenon.

Particularly useful sequencing reactions are Sequencing By Binding™(SBB™) reactions as described in commonly owned US Pat. App. Pub. No.2017/0022553 A1; U.S. Pat. App. Ser. No. 62/447,319 to which US Pat App.Pub. No. 2018/0044727 A1 claims priority; 62/440,624 to which US PatApp. Pub. No. 2018/0187245 A1 claims priority; or 62/450,397 to which USPat App. Pub. No. 2018/0208983 A1 claims priority, each of which isincorporated herein by reference. Generally, methods for determining thesequence of a template nucleic acid molecule can be based on formationof a ternary complex (between polymerase, primed nucleic acid andcognate nucleotide) under specified conditions. The method can includean examination phase followed by a nucleotide incorporation phase.

The examination phase can be carried out in a flow cell (or othervessel), the flow cell containing at least one template nucleic acidmolecule primed with a primer by delivering to the flow cell reagents toform a first reaction mixture. The reaction mixture can include theprimed template nucleic acid, a polymerase and at least one nucleotidetype. Interaction of polymerase and a nucleotide with the primedtemplate nucleic acid molecule(s) can be observed under conditions wherethe nucleotide is not covalently added to the primer(s); and the nextbase in each template nucleic acid can be identified using the observedinteraction of the polymerase and nucleotide with the primed templatenucleic acid molecule(s). The interaction between the primed template,polymerase and nucleotide can be detected in a variety of schemes. Forexample, the nucleotides can contain a detectable label. Each nucleotidecan have a distinguishable label with respect to other nucleotides.Alternatively, some or all of the different nucleotide types can havethe same label and the nucleotide types can be distinguished based onseparate deliveries of different nucleotide types to the flow cell. Insome embodiments, the polymerase can be labeled. Polymerases that areassociated with different nucleotide types can have unique labels thatdistinguish the type of nucleotide to which they are associated.Alternatively, polymerases can have similar labels and the differentnucleotide types can be distinguished based on separate deliveries ofdifferent nucleotide types to the flow cell. Detection can be carriedout by scanning the flow cell using an apparatus or method set forthherein.

During the examination phase, discrimination between correct andincorrect nucleotides can be facilitated by ternary complexstabilization. A variety of conditions and reagents can be useful. Forexample, the primer can contain a reversible blocking moiety thatprevents covalent attachment of nucleotide; and/or cofactors that arerequired for extension, such as divalent metal ions, can be absent;and/or inhibitory divalent cations that inhibit polymerase-based primerextension can be present; and/or the polymerase that is present in theexamination phase can have a chemical modification and/or mutation thatinhibits primer extension; and/or the nucleotides can have chemicalmodifications that inhibit incorporation, such as 5′ modifications thatremove or alter the native triphosphate moiety. The examination phasecan include scanning of the flow cell using apparatus and methods setforth herein.

The extension phase can then be carried out by creating conditions inthe flow cell where a nucleotide can be added to the primer on eachtemplate nucleic acid molecule. In some embodiments, this involvesremoval of reagents used in the examination phase and replacing themwith reagents that facilitate extension. For example, examinationreagents can be replaced with a polymerase and nucleotide(s) that arecapable of extension. Alternatively, one or more reagents can be addedto the examination phase reaction to create extension conditions. Forexample, catalytic divalent cations can be added to an examinationmixture that was deficient in the cations, and/or polymerase inhibitorscan be removed or disabled, and/or extension competent nucleotides canbe added, and/or a deblocking reagent can be added to render primer(s)extension competent, and/or extension competent polymerase can be added.

It will be understood that any of a variety of nucleic acid sequencingreactions can be carried out using an apparatus and method of thepresent disclosure. Other exemplary sequencing methods are set forthbelow.

Sequencing-by-synthesis (SBS) techniques can be used. SBS generallyinvolves the enzymatic extension of a nascent primer through theiterative addition of nucleotides against a template strand to which theprimer is hybridized. Briefly, SBS can be initiated by contacting targetnucleic acids, attached to sites in a vessel, with one or more labelednucleotides, DNA polymerase, etc. Those sites where a primer is extendedusing the target nucleic acid as template will incorporate a labelednucleotide that can be detected. Detection can include scanning using anapparatus or method set forth herein. Optionally, the labelednucleotides can further include a reversible termination property thatterminates further primer extension once a nucleotide has been added toa primer. For example, a nucleotide analog having a reversibleterminator moiety can be added to a primer such that subsequentextension cannot occur until a deblocking agent is delivered to removethe moiety. Thus, for embodiments that use reversible termination, adeblocking reagent can be delivered to the vessel (before or afterdetection occurs). Washes can be carried out between the variousdelivery steps. The cycle can be performed n times to extend the primerby n nucleotides, thereby detecting a sequence of length n. ExemplarySBS procedures, reagents and detection components that can be readilyadapted for use with a detection apparatus produced by the methods ofthe present disclosure are described, for example, in Bentley et al.,Nature 456:53-59 (2008), WO 04/018497; WO 91/06678; WO 07/123744; U.S.Pat. Nos. 7,057,026; 7,329,492; 7,211,414; 7,315,019 or 7,405,281, andUS Pat. App. Pub. No. 2008/0108082 A1, each of which is incorporatedherein by reference. Also useful are SBS methods that are commerciallyavailable from Illumina, Inc. (San Diego, Calif.).

Some SBS embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use reagents andan electrical detector that are commercially available from ThermoFisher(Waltham, Mass.) or described in US Pat. App. Pub. Nos. 2009/0026082 A1;2009/0127589 A1; 2010/0137143 A1; or 2010/0282617 A1, each of which isincorporated herein by reference.

Other sequencing procedures can be used, such as pyrosequencing.Pyrosequencing detects the release of inorganic pyrophosphate (PPi) asnucleotides are incorporated into a nascent primer hybridized to atemplate nucleic acid strand (Ronaghi, et al., Analytical Biochemistry242 (1), 84-9 (1996); Ronaghi, Genome Res. 11 (1), 3-11 (2001); Ronaghiet al. Science 281 (5375), 363 (1998); U.S. Pat. Nos. 6,210,891;6,258,568 and 6,274,320, each of which is incorporated herein byreference). In pyrosequencing, released PPi can be detected by beingconverted to adenosine triphosphate (ATP) by ATP sulfurylase, and theresulting ATP can be detected via luciferase-produced photons. Thus, thesequencing reaction can be monitored via a luminescence detection systemthat is configured to scan a vessel using apparatus and methods setforth herein.

Sequencing-by-ligation reactions are also useful including, for example,those described in Shendure et al. Science 309:1728-1732 (2005); U.S.Pat. No. 5,599,675; or U.S. Pat. No. 5,750,341, each of which isincorporated herein by reference. Some embodiments can includesequencing-by-hybridization procedures as described, for example, inBains et al., Journal of Theoretical Biology 135 (3), 303-7 (1988);Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al.,Science 251 (4995), 767-773 (1995); or WO 1989/10977, each of which isincorporated herein by reference. In both sequencing-by-ligation andsequencing-by-hybridization procedures, primers that are hybridized tonucleic acid templates are subjected to repeated cycles of extension byoligonucleotide ligation. Typically, the oligonucleotides arefluorescently labeled and can be detected to determine the sequence ofthe template, for example, using a scanning apparatus or method setforth herein.

Some embodiments can utilize methods involving real-time monitoring ofDNA polymerase activity. For example, nucleotide incorporations can bedetected through fluorescence resonance energy transfer (FRET)interactions between a fluorophore-bearing polymerase andgamma-phosphate-labeled nucleotides, or with zero-mode waveguides (ZMW).Techniques and reagents for sequencing via FRET and or ZMW detectionthat can be modified for use in an apparatus or method set forth hereinare described, for example, in Levene et al. Science 299, 682-686(2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); Korlach et al.Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008); or U.S. Pat. Nos.7,315,019; 8,252,911 or 8,530,164, the disclosures of which areincorporated herein by reference.

Steps for the above sequencing methods can be carried out cyclically.For example, examination and extension steps of an SBB™ method can berepeated such that in each cycle a single next correct nucleotide isexamined (i.e. the next correct nucleotide being a nucleotide thatcorrectly binds to the nucleotide in a template nucleic acid that islocated immediately 5′ of the base in the template that is hybridized tothe 3′-end of the hybridized primer) and, subsequently, a single nextcorrect nucleotide is added to the primer. Any number of cycles of asequencing method set forth herein can be carried out including, forexample, at least 1, 2, 5, 10, 20, 25, 30, 40, 50, 75, 100, 150 or morecycles. Alternatively or additionally, no more than 150, 100, 75, 50,40, 30, 25, 20, 10, 5, 2 or 1 cycles are carried out.

Nucleic acid template(s), to be sequenced, can be added to a vesselusing any of a variety of known methods. In some embodiments, a singlenucleic acid molecule is to be sequenced. The nucleic acid molecule canbe delivered to a vessel and can optionally be attached to a surface inthe vessel. In some embodiments, the molecule is subjected to singlemolecule sequencing. Alternatively, multiple copies of the nucleic acidcan be made and the resulting ensemble can be sequenced. For example,the nucleic acid can be amplified on a surface (e.g. on the inner wallof a flow cell) using techniques set forth in further detail below.

In multiplex embodiments, a variety of different nucleic acid molecules(i.e. a population having a variety of different sequences) aresequenced. The molecules can optionally be attached to a surface in avessel. The nucleic acids can be attached at unique sites on the surfaceand single nucleic acid molecules that are spatially distinguishable onefrom the other can be sequenced in parallel. Alternatively, the nucleicacids can be amplified on the surface to produce a plurality of surfaceattached ensembles. The ensembles can be spatially distinguishable andsequenced in parallel.

A method set forth herein can use any of a variety of amplificationtechniques in a vessel. Exemplary techniques that can be used include,but are not limited to, polymerase chain reaction (PCR), rolling circleamplification (RCA), multiple displacement amplification (MDA), bridgeamplification, or random prime amplification (RPA). In particularembodiments, one or more primers used for amplification can be attachedto a surface in a vessel. In such embodiments, extension of thesurface-attached primers along template nucleic acids will result incopies of the templates being attached to the surface. Methods thatresult in one or more sites on a solid support, where each site isattached to multiple copies of a particular nucleic acid template, canbe referred to as “clustering” methods.

In PCR embodiments, one or both primers used for amplification can beattached to a surface. Formats that utilize two species of attachedprimer are often referred to as bridge amplification because doublestranded amplicons form a bridge-like structure between the two attachedprimers that flank the template sequence that has been copied. Exemplaryreagents and conditions that can be used for bridge amplification aredescribed, for example, in U.S. Pat. Nos. 5,641,658 or U.S. Pat. No.7,115,400; U.S. Patent Pub. Nos. 2002/0055100 A1, 2004/0096853 A1,2004/0002090 A1, 2007/0128624 A1 or 2008/0009420 A1, each of which isincorporated herein by reference. PCR amplification can also be carriedout with one of the amplification primers attached to the surface andthe second primer in solution. An exemplary format that uses acombination of one solid phase-attached primer and a solution phaseprimer is known as primer walking and can be carried out as described inU.S. Pat. No. 9,476,080, which is incorporated herein by reference.Another example is emulsion PCR which can be carried out as described,for example, in Dressman et al., Proc. Natl. Acad. Sci. USA100:8817-8822 (2003), WO 05/010145, or U.S. Patent Pub. Nos.2005/0130173 A1 or 2005/0064460 A1, each of which is incorporated hereinby reference.

RCA techniques can be used in a method set forth herein. Exemplaryreagents that can be used in an RCA reaction and principles by which RCAproduces amplicons are described, for example, in Lizardi et al., Nat.Genet. 19:225-232 (1998) or US Pat. App. Pub. No. 2007/0099208 A1, eachof which is incorporated herein by reference. Primers used for RCA canbe in solution or attached to a surface in a flow cell.

MDA techniques can also be used in a method of the present disclosure.Some reagents and useful conditions for MDA are described, for example,in Dean et al., Proc Natl. Acad. Sci. USA 99:5261-66 (2002); Lage etal., Genome Research 13:294-307 (2003); Walker et al., Molecular Methodsfor Virus Detection, Academic Press, Inc., 1995; Walker et al., Nucl.Acids Res. 20:1691-96 (1992); or U.S. Pat. Nos. 5,455,166; 5,130,238; or6,214,587, each of which is incorporated herein by reference. Primersused for MDA can be in solution or attached to a surface in a vessel.

In particular embodiments, a combination of the above-exemplifiedamplification techniques can be used. For example, RCA and MDA can beused in a combination wherein RCA is used to generate a concatemericamplicon in solution (e.g. using solution-phase primers). The ampliconcan then be used as a template for MDA using primers that are attachedto a surface in a vessel. In this example, amplicons produced after thecombined RCA and MDA steps will be attached in the vessel. The ampliconswill generally contain concatemeric repeats of a target nucleotidesequence.

Nucleic acid templates that are used in a method or composition hereincan be DNA such as genomic DNA, synthetic DNA, amplified DNA,complementary DNA (cDNA) or the like. RNA can also be used such as mRNA,ribosomal RNA, tRNA or the like. Nucleic acid analogs can also be usedas templates herein. Thus, a mixture of nucleic acids used herein can bederived from a biological source, synthetic source or amplificationproduct. Primers used herein can be DNA, RNA or analogs thereof.

Exemplary organisms from which nucleic acids can be derived include, forexample, those from a mammal such as a rodent, mouse, rat, rabbit,guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate,human or non-human primate; a plant such as Arabidopsis thaliana, corn,sorghum, oat, wheat, rice, canola, or soybean; an algae such asChlamydomonas reinhardtii; a nematode such as Caenorhabditis elegans; aninsect such as Drosophila melanogaster, mosquito, fruit fly, honey beeor spider; a fish such as zebrafish; a reptile; an amphibian such as afrog or Xenopus laevis; a dictyostelium discoideum; a fungi such aspneumocystis carinii, Takifugu rubripes, yeast, Saccharamoycescerevisiae or Schizosaccharomyces pombe; or a plasmodium falciparum.Nucleic acids can also be derived from a prokaryote such as a bacterium,Escherichia coli, staphylococci or mycoplasma pneumoniae; an archae; avirus such as Hepatitis C virus or human immunodeficiency virus; or aviroid. Nucleic acids can be derived from a homogeneous culture orpopulation of the above organisms or alternatively from a collection ofseveral different organisms, for example, in a community or ecosystem.Nucleic acids can be isolated using methods known in the art including,for example, those described in Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York(2001) or in Ausubel et al., Current Protocols in Molecular Biology,John Wiley and Sons, Baltimore, Md. (1998), each of which isincorporated herein by reference. Cells, tissues, biological fluids,proteins and other samples can be obtained from these organisms anddetected using an apparatus or method set forth herein.

A template nucleic acid can be obtained from a preparative method suchas genome isolation, genome fragmentation, gene cloning and/oramplification. The template can be obtained from an amplificationtechnique such as polymerase chain reaction (PCR), rolling circleamplification (RCA), multiple displacement amplification (MDA) or thelike. Exemplary methods for isolating, amplifying and fragmentingnucleic acids to produce templates for analysis on an array are setforth in U.S. Pat. No. 6,355,431 or U.S. Pat. No. 9,045,796, each ofwhich is incorporated herein by reference. Amplification can also becarried out using a method set forth in Sambrook et al., MolecularCloning: A Laboratory Manual, 3rd edition, Cold Spring HarborLaboratory, New York (2001) or in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1998), each ofwhich is incorporated herein by reference.

The present disclosure further provides a detection apparatus thatincludes (a) a vessel having a lumen and a wall, wherein the wall has aninternal surface and an external surface, wherein the wall has aplurality of discrete contacts between the internal surface and theexternal surface, wherein the internal surface contacts the lumen, andwherein the plurality of discrete contacts occupies a length l in a scandimension x; (b) a transmissive surface; (c) a preload configured tourge discrete contacts on the external surface of the vessel to contactthe transmissive surface, optionally, the area of the transmissivesurface can have a maximum length in the scan dimension x that isshorter than length l; (d) a scan actuator configured to slide thevessel along the transmissive surface in the scan dimension x; and (e) adetector configured to acquire signals from the discrete contacts viathe transmissive surface.

As exemplified in several embodiments herein, optical signals can berelayed to a detection apparatus via transmissive surface that istransparent to optical signals. An objective serves as a usefultransmitter of optical signals from a vessel to a detector. In someembodiments the transmitter is an array of lenses. The lenses in thearray can be configured to collect signals from (or direct energy to)different areas in an xy plane. The lenses can be arranged to collectsignals from contiguous areas in the xy plane or, alternatively, theareas that are observed can be separated by interstitial regions thatare not observed when the areas are observed. In some embodiments, thevessel includes an array of sites that is configured to be observed byan array of lenses. Each lens can be configured to simultaneouslyobserve one or more sites in the array of sites. For example, each lenscan be configured to observe at least 1, 4, 9, 16, 25, 36, 49, 64, 81,100 or more sites in an array of sites. Alternatively or additionally,each lens can be configured to observe at most 100, 81, 64, 49, 36, 25,16, 9, 4 or 1 site(s) in an array of sites. Accordingly, an embodimentis provided wherein each lens is configured to observe a single site.

Each lens in an array of lenses can be aligned with its own opticaltrain to direct radiation to one or more detector. Alternatively,multiple lenses can be combined into a common optical train to directradiation to one or more detector. The optical trains can include any ofa variety of optical components including, but not limited to, acollimating lens for collimating signals from the array of sites, acolor separating element for spectrally separating radiation; and afocusing lens for focusing radiation from the sites to a detector.Exemplary configurations for an array of lenses and an array of sitesobserved by the lenses is provides in U.S. Pat. No. 9,581,550, which isincorporated herein by reference. For example, the sites of the arraycan be zero mode waveguides (ZMWs).

Other transmitters can be used as appropriate for the energy or signalthat is to be transmitted. For example, a transmissive surface canconduct electrical signals, thermal signals, magnetic signals, pressuresignals, audio signals, or the like. Temporary electrical contacts suchas pogo pins can be used to transmit electrical signals between thetransmissive surface and vessel. A transmitter that is present in anapparatus set forth herein can transmit energy of a variety of forms,including but not limited to the aforementioned signals.

In a particular embodiment, the transmissive surface or the internalsurface of the vessel includes an electronic detector such as afield-effect transistor (FET) or complementary metal oxide semiconductor(CMOS). Particularly useful electronic detectors include, for example,those used for nucleic acid sequencing applications such as those usedfor detection of protons as set forth in US Pat. App. Pub. Nos.2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; or 2010/0282617 A1,each of which is incorporated herein by reference. Also useful areelectronic detectors used to detect optical signals including forexample, those set forth in US Pat. App. Pub. Nos. 2009/0197326 A1;2015/0293021 A1; 2016/0017416 A1; or 2016/0356715 A1, each of which isincorporated herein by reference.

The apparatus and methods of the present disclosure have beenexemplified in the context of use for nucleic acid sequencing reactions.The apparatus and methods can be used for other analytical applicationsas well. Generally, analytical applications that are carried out inscanning microscopes can be applied to apparatus and methods of thepresent disclosure. For example, the methods or apparatus can beconfigured to scan microarrays that are used for analyzing enzymeactivity, binding of ligands to receptors, binding of complementarynucleic acids to each other, presence of mutations (such as singlenucleotide polymorphisms (SNPs)) in nucleic acids, expression level forRNA species. Microarrays that are detected via optical labels, such asfluorophores, are particularly applicable. Larger biological samplessuch as cells or tissues can be detected using a method or apparatusherein. Again, detection modalities that utilize optically detectedprobes or stains are particularly applicable. Other uses includeevaluation of manufactured products for which quality or othercharacteristics are evaluated via microscopic scanning. Exemplaryproducts include, but are not limited to, computer chips, sensors,electronic components and other devices that are microfabricated ornanofabricated. Tests known in the art of molecular diagnostics can bemodified for use in an apparatus or method set forth herein such asbinding assays (e.g. enzyme-linked immunosorbent assay (ELISA)), realtime polymerase chain reaction assays and the like.

Apparatus and methods set forth herein in the context of detectingreactions can be readily modified for use in preparative methods. Inparticular embodiments, the present disclosure provides reactorapparatus. A reactor apparatus can include (a) a vessel having a lumenand a wall, wherein the wall has an internal surface and an externalsurface, wherein the internal surface contacts the lumen; (b) areference surface that forms a structural loop with an energy source;(c) a preload configured to urge the external surface of the vessel tocontact an area on the reference surface; (d) a scan actuator configuredto slide the vessel along the reference surface in a scan dimension; and(e) a transmitter configured to direct energy from the energy source tothe internal surface or the lumen when the external surface of thevessel is urged by the preload to contact the reference surface.

Also provided is a method of performing reactions in a vessel. Themethod can include (a) translating a vessel along a reference surface ofa reactor apparatus, wherein the vessel comprises a lumen and a wall,wherein the lumen comprises reactants, wherein the reference surfacecontacts at least a portion of the vessel during the translating, andwherein the reference surface forms a structural loop with an energysource; and (b) directing energy from the energy source to the reactantsat different locations along the vessel, wherein the vessel is urged tothe reference surface by a preload during the directing of the energy tothe reactants, thereby performing reactions in the vessel.

A method of performing reactions can include (a) delivering energy froma reactor apparatus to a first subset of reactants in a vessel whileapplying a preload to a first portion of the vessel, wherein the preloadpositions the first subset of reactants to occupy an xy plane of areaction zone, wherein the preload is not applied to a second portion ofthe vessel; (b) translating the vessel to position a second subset ofthe reactants in the xy plane of the reaction zone; and (c) deliveringenergy from the reactor apparatus to the second subset of the analytesin the vessel while applying the preload to a second portion of thevessel, wherein the preload positions the second subset of the analytesto occupy the xy plane, wherein the preload is not applied to the firstportion of the vessel, thereby performing reactions in the vessel.

Exemplary energy sources that can be used in apparatus herein include,but are not limited to, radiation sources such as a laser, lightemitting diode (LED), lamp, microwave source, or x-ray generator;electricity source; ion beam source such as a duoplasmitron; electronemitter such as a hot filament or hollow cathode; electric currentsource; or voltage source.

Throughout this application various publications, patents and/or patentapplications have been referenced. The disclosures of these documents intheir entireties are hereby incorporated by reference in thisapplication.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

1-25. (canceled)
 26. A method of scanning a vessel, comprising (a)examining a first subset of analytes in a vessel while applying apreload to a first portion of the vessel, wherein the preload positionsthe first subset of analytes to occupy an xy plane in a detection zone,wherein the preload is not applied to a second portion of the vessel;(b) translating the vessel to position a second subset of the analytesin the xy plane of the detection zone; and (c) examining the secondsubset of the analytes in the vessel while applying the preload to thesecond portion of the vessel, wherein the preload positions the secondsubset of the analytes to occupy the xy plane of the detection zone,wherein the preload is not applied to the first portion of the vessel,thereby scanning the vessel.
 27. The method of claim 26, wherein thepreload is configured to urge the vessel to contact a reference surface.28. The method of claim 27, wherein the reference surface contacts onlya portion of the vessel while the vessel is examined.
 29. The method ofclaim 26, wherein the preload acts in opposition to pressurized gas toposition the vessel while the vessel is examined.
 30. The method ofclaim 26, wherein the preload acts in opposition to pressurized liquidto position the vessel while the vessel is examined.
 31. The method ofclaim 26, wherein the preload acts in opposition to a magnetic field toposition the vessel while the vessel is examined.
 32. The method ofclaim 26, wherein the preload acts in opposition to an electric field toposition the vessel while the vessel is examined.
 33. The method ofclaim 26, wherein the vessel comprises a lumen and a wall, wherein theinternal surface contacts the lumen.
 34. The method of claim 33, whereinthe first subset of analytes is arrayed on the internal surface of thevessel.
 35. The method of claim 34, wherein the first subset of analytesis within the lumen of the vessel.
 36. The method of claim 26, whereinthe examining comprises acquiring optical signals produced by theanalytes.
 37. The method of claim 36, wherein the detection apparatusfurther comprises an objective that is configured to transmit opticalsignals from the vessel to a camera.
 38. The method of claim 37, furthercomprising focusing the objective by changing the relative distancebetween the objective and the preload in a direction that is orthogonalto the xy plane.
 39. The method of claim 26, wherein the translatingcomprises contacting a rotating gear with perforations on the vessel oron a cartridge that holds the vessel.
 40. The method of claim 26,wherein the translating comprises magnetic interactions of a linearactuator with a magnetic or ferromagnetic material on the vessel or on acartridge that holds the vessel.
 41. The method of claim 26, wherein thevessel is urged by the preload during the translating.
 42. The method ofclaim 26, wherein the scanning of the vessel is carried out in a methodof sequencing nucleic acids.
 43. The method of claim 42, wherein themethod of sequencing further comprises delivering polymerases andnucleotides to the vessel.
 44. The method of claim 43, wherein thepolymerases and nucleotides are delivered in a plurality of repeatedcycles, wherein the repeated cycles comprise the scanning of the vessel.45. The method of claim 42, wherein the sites are labeled withfluorescent labels during the method of sequencing.
 46. The method ofclaim 45, wherein the fluorescent labels are excited via radiationpassing through the objective and wherein fluorescent emission from thelabels is detected through the objective.
 47. The method of claim 26,wherein the examining is carried out using an array of lenses that isconfigured to transmit optical signals from an array of sites in thevessel to a detector.
 48. The method of claim 47, wherein each lens inthe array of lenses is configured to observe a single site in an arrayof sites.